U.S. patent application number 14/736734 was filed with the patent office on 2015-12-17 for systems and methods for substrate enrichment.
The applicant listed for this patent is LIFE TECHNOLOGIES CORPORATION. Invention is credited to Brian REED.
Application Number | 20150361418 14/736734 |
Document ID | / |
Family ID | 54835644 |
Filed Date | 2015-12-17 |
United States Patent
Application |
20150361418 |
Kind Code |
A1 |
REED; Brian |
December 17, 2015 |
Systems and Methods for Substrate Enrichment
Abstract
A method of separating bead substrates includes applying an
emulsion to an emulsion-breaking solution. A dispersed phase of the
emulsion includes an unbound polynucleotide, a first set of bead
substrates and a second set of bead substrates. The unbound
polynucleotide includes a segment complementary to a coupling
oligonucleotide. The first set of bead substrates includes the
coupling oligonucleotide extended to include a segment
complementary to a portion of the unbound polynucleotide. The
second set of bead substrates includes the coupling
oligonucleotide. The emulsion-breaking solution includes an
interference probe having a sequence similar to the coupling
oligonucleotide or complementary to the coupling oligonucleotide.
The method further includes binding beads of the first set of bead
substrates to separation substrates and separating unbound beads of
the second set of bead substrates from the beads of the first set
of bead substrates bound to the separation substrates.
Inventors: |
REED; Brian; (Woodbridge,
CT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LIFE TECHNOLOGIES CORPORATION |
Carlsbad |
CA |
US |
|
|
Family ID: |
54835644 |
Appl. No.: |
14/736734 |
Filed: |
June 11, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62010573 |
Jun 11, 2014 |
|
|
|
Current U.S.
Class: |
536/25.4 |
Current CPC
Class: |
C07H 1/06 20130101; C12Q
2565/519 20130101; C12Q 2563/157 20130101; C12Q 2563/143 20130101;
C12Q 1/6844 20130101; C12Q 1/6806 20130101; C12Q 1/6844 20130101;
C12N 15/1013 20130101 |
International
Class: |
C12N 15/10 20060101
C12N015/10; C12Q 1/68 20060101 C12Q001/68 |
Claims
1. A method of separating bead substrates, the method comprising:
applying an emulsion to an emulsion-breaking solution, a dispersed
phase of the emulsion comprising an unbound polynucleotide, a first
set of bead substrates and a second set of bead substrates, the
unbound polynucleotide including a segment complementary to a
coupling oligonucleotide, the first set of bead substrates
including the coupling oligonucleotide extended to include a
segment complementary to a portion of the unbound polynucleotide,
the second set of bead substrates including the coupling
oligonucleotide, the emulsion-breaking solution including an
interference probe having a sequence similar to the coupling
oligonucleotide or complementary to the coupling oligonucleotide;
binding beads of the first set of bead substrates to separation
substrates; and separating unbound beads of the second set of bead
substrates from the beads of the first set of bead substrates bound
to the separation substrates.
2. The method of claim 1, wherein the interference probe has the
sequence similar to the coupling oligonucleotide.
3. The method of claim 1, wherein the interference probe has a
sequence complementary to the coupling oligonucleotide.
4. The method of claim 1, wherein beads of the second set of bead
substrates are substantially free of extension to the coupling
oligonucleotide.
5. The method of claim 1, wherein the unbound polynucleotide
includes a capture functionality.
6. The method of claim 5, wherein the extended coupling
oligonucleotide of the first set of bead substrates is hybridized
to a polynucleotide including the capture functionality.
7. The method of claim 6, wherein the coupling functionality
includes biotin.
8. The method of claim 6, wherein the separation substrates include
functionality to attach to the capture functionality.
9. The method of claim 1, wherein the separation substrates include
magnetic beads.
10. The method of claim 9, further comprising, prior to separating
the unbound beads of the second set of bead substrates,
immobilizing with a magnet the magnetic beads bound to beads of the
first set of bead substrates.
11. The method of claim 1, wherein the emulsion-breaking solution
includes a surfactant.
12. The method of claim 11, wherein the surfactant includes a
non-ionic surfactant.
13. The method of claim 11, wherein the surfactant includes an
ionic surfactant.
14. The method of claim 13, wherein the ionic surfactant is an
anionic surfactant.
15. A method of separating bead substrates, the method comprising:
applying an emulsion to an emulsion-breaking solution, a dispersed
phase of the emulsion comprising an unbound polynucleotide, a first
set of bead substrates and a second set of bead substrates, the
unbound polynucleotide including a first segment complementary to a
coupling oligonucleotide and a second segment attached to a capture
functionality, the first set of bead substrates including the
coupling oligonucleotide hybridized to a polynucleotide including
the first segment complementary to the coupling nucleotide and the
second segment attached to the capture functionality, the second
set of bead substrates including the coupling oligonucleotide
substantially not hybridized to the polynucleotide or the unbound
polynucleotide, the emulsion-breaking solution including an
interference probe having a sequence similar to the coupling
oligonucleotide or complementary to the coupling oligonucleotide;
binding beads of the first set of bead substrates to separation
substrates, the separation substrates including functionality to
attach to the capture functionality; and separating unbound beads
of the second set of bead substrates from the bound beads of the
first set of bead substrates.
16. The method of claim 15, wherein the interference probe has the
sequence similar to the coupling oligonucleotide.
17. The method of claim 15, wherein the interference probe has a
sequence complementary to the coupling oligonucleotide.
18. The method of claim 15, wherein the coupling functionality
includes biotin and the functionality to attach to the capture
functionality includes streptavidin.
19. The method of claim 15, wherein the separation substrates
include magnetic beads.
20. The method of claim 19, further comprising, prior to separating
the unbound beads of the second set of bead substrates,
immobilizing with a magnet the magnetic beads bound to beads of the
first set of bead substrates.
Description
CROSS-REFERENCE TO RELATED APPLICATION(S)
[0001] This application claims benefit of U.S. Provisional
Application No. 61/010,573, filed Jun. 11, 2014, which is
incorporated herein by reference in its entirety.
FIELD OF THE DISCLOSURE
[0002] This disclosure, in general, relates to systems and methods
for enriching substrates suspended in a dispersion.
BACKGROUND
[0003] As biological and chemical sciences advance, scientists
increasingly turn to separation techniques to isolate and analyze
analytes. In particular, the chemical sciences are turning to
enrichment methods to isolate and analyze ever smaller
concentrations of compounds. In the biological sciences, scientists
seek to isolate and analyze increasingly complex molecules, such as
DNA, RNA, and proteins, such as enzymes.
[0004] In particular, compounds can be bound to dispersed
substrates, such as particles or beads, and then isolated for
analysis. In an example, a substrate bound to compounds can be
applied to a sensor for analysis. In another example, the compounds
can be separated from the substrates following isolation and can be
analyzed separately. Such isolated substrates bound to compounds or
analytes can be used for detecting trace amounts of chemical
agents, biologically active reagents, or can be used in analyzing
molecules, such as DNA or RNA. In particular, such dispersed
substrates bound to polynucleotides or proteins can be used for
sequencing.
SUMMARY
[0005] In an exemplary embodiment, amplified substrates are
conjugated with copies of a target sequence through emulsion
amplification. The emulsion is applied over a breaking solution
that includes interference probes that limit binding of unbound
template polynucleotides to unamplified substrates that lack
conjugation to the copies of the target sequence. The amplified
substrates are separated from the unamplified substrates, thus
enriching the amplified substrates. The enriched amplified
substrates can be loaded onto biosensors.
BRIEF DESCRIPTION OF THE DRAWINGS
[0006] The present disclosure may be better understood, and its
numerous features and advantages made apparent to those skilled in
the art by referencing the accompanying drawings.
[0007] FIG. 1 includes a block flow diagram of an exemplary method
for analyzing a target polynucleotide.
[0008] FIG. 2 includes a pictorial illustration of an exemplary
method for analyzing a target polynucleotide.
[0009] FIG. 3 and FIG. 4 include pictorial illustrations of
exemplary methods for amplifying a target polynucleotide.
[0010] FIG. 5 includes an illustration of an exemplary
emulsion.
[0011] FIG. 6 includes a pictorial illustration of exemplary
byproducts following emulsion amplification.
[0012] FIG. 7 includes a block flow diagram illustrating an
exemplary method for enriching dispersed substrates.
[0013] FIG. 8 and FIG. 9 include illustrations of exemplary devices
for enriching dispersed substrates.
[0014] FIG. 10 includes an illustration of exemplary system for
sequencing.
DESCRIPTION OF THE PREFERRED EMBODIMENT(S)
[0015] In an exemplary embodiment, substrates, such as bead or
particle substrates, are amplified to include multiple copies of
target polynucleotides using an emulsion. Herein, amplified
substrates are substrates to which copies of a target
polynucleotide are attached. For example, an amplification reaction
can extend primers conjugated to the substrate. Such primers are
referred to herein as coupling oligonucleotides or coupling sites.
Following amplification, the emulsion can be applied over a
breaking solution that includes interference probes to prevent
amplification byproducts from binding to unamplified substrates.
Amplified substrates are separated from the unamplified substrates,
and the amplified substrates are loaded onto a biosensor. The
biosensor loaded with the amplified substrates can be used to
sequence the target polynucleotides or perform other testing. The
amplification reaction can utilize polymerase chain reaction (PCR)
or can utilize recombinase polymerase amplification (RPA). In an
example, the interference probe is complementary to a coupling
oligonucleotide coupled to the substrates. In another example, the
interference probe has the same sequence as the coupling
oligonucleotide and can be complementary to a segment of an unbound
template polynucleotide. In a particular example, amplified
substrates can be bound to a magnetic substrate and the amplified
substrates can be separated from unamplified substrates utilizing
magnetic separation techniques.
[0016] As illustrated in FIG. 1, a method 100 includes combining
substrates, amplification reagents, and target polynucleotides into
an amplification solution, as illustrated at 102. The substrates
can include bead or particulate substrates. In particular, a
substrate includes bead substrates formed of hydrophilic polymers.
The substrates can carry a charge. Alternatively, the substrates
can be neutral.
[0017] For example, substrates can be formed from monomers
including a radically polymerizable monomer, such as a vinyl-based
monomer. In an example, the monomer can include acrylamide, vinyl
acetate, hydroxyalkylmethacrylate, or any combination thereof. In a
particular example, the hydrophilic monomer is an acrylamide, such
as an acrylamide including hydroxyl groups, amino groups, carboxyl
groups, or a combination thereof. In an example, the hydrophilic
monomer is an aminoalkyl acrylamide, an acrylamide functionalized
with an amine terminated polypropylene glycol (D, illustrated
below), an acrylopiperazine (C, illustrated below), or a
combination thereof. In another example, the acrylamide can be a
hydroxyalkyl acrylamide, such as hydroxyethyl acrylamide. In
particular, the hydroxyalkyl acrylamide can include
N-tris(hydroxymethyl)methyl)acrylamide (A, illustrated below),
N-(hydroxymethyl)acrylamide (B, illustrated below), or a
combination thereof. In a further example, a mixture of monomers,
such as a mixture of hydroxyalkyl acrylamide and amine
functionalize acrylamide or a mixture of acrylamide and amine
functionalized acrylamide, can be used. In an example, the amine
functionalize acrylamide can be included in a ratio of hydroxyalkyl
acrylamide:amine functionalized acrylamide or acrylamide:amine
functionalized acrylamide in a range of 100:1 to 1:1, such as a
range of 100:1 to 2:1, a range of 50:1 to 3:1, a range of 50:1 to
5:1 or even a range of 50:1 to 10:1.
##STR00001##
[0018] In a particular example, the substrates are hydrogel bead
substrates.
[0019] Each of the substrates can include coupling sites to which a
template polynucleotide can hybridize. For example, the coupling
sites can each include a coupling oligonucleotide complementary to
a section of a template polynucleotide. The template polynucleotide
can include the target polynucleotide or segments complementary to
the target polynucleotide, in addition to segments complementary to
the coupling oligonucleotide.
[0020] The coupling oligonucleotide can be conjugated to the
substrate. The polymer of a substrate can be activated to
facilitate conjugation with a polynucleotide or oligonucleotide,
such as a coupling oligonucleotide. For example, functional groups
on the substrate can be enhanced to permit binding with
polynucleotides or oligonucleotides. In a particular example,
functional groups of the hydrophilic polymer can be modified with
reagents capable of converting the hydrophilic polymer functional
groups to reactive moieties that can undergo nucleophilic or
electrophilic substitution. For example, hydroxyl groups on the
substrate can be activated by replacing at least a portion of the
hydroxyl groups with a sulfonate group or chlorine. Exemplary
sulfonate groups can be derived from tresyl, mesyl, tosyl, or fosyl
chloride, or any combination thereof. Sulfonate can act to permit
nucleophiles to replace the sulfonate. The sulfonate may further
react with liberated chlorine to provide chlorinated groups that
can be used in a process to conjugate the particles. In another
example, amine groups on a substrate can be activated.
[0021] For example, polynucleotides or oligonucleotides can bind to
the hydrophilic polymer through nucleophilic substitution with the
sulfonate group. In particular example, polynucleotides or
oligonucleotides terminated with a nucleophile, such as an amine or
a thiol, can undergo nucleophilic substitution to replace the
sulfonate groups on the surface of the substrate.
[0022] In another example, sulfonated substrates can be further
reacted with mono- or multi-functional mono- or multi-nucleophilic
reagents that can form an attachment to the particle while
maintaining nucleophilic activity for oligonucleotides comprising
electrophilic groups, such as maleimide. In addition, the residual
nucleophilic activity can be converted to electrophilic activity by
attachment to reagents comprising multi-electrophilic groups, which
are subsequently to attach to polynucleotides or oligonucleotides
terminated with nucleophilic groups.
[0023] In another example, a monomer containing the functional
group can be added during the polymerization. The monomer can
include, for example, an acrylamide containing a carboxylic acid,
ester, halogen or other amine reactive group. The ester group may
be hydrolyzed before the reaction with an amine terminated
polynucleotide or oligonucleotide.
[0024] Other conjugation techniques include the use of monomers
that comprise amines. The amine is a nucleophilic group that can be
further modified with amine reactive bi-functional
bis-electrophilic reagents that yield a mono-functional
electrophilic group subsequent to attachment to the substrate. Such
an electrophilic group can be reacted with polynucleotides or
oligonucleotides having a nucleophilic group, such as an amine or
thiol, causing attachment of the oligonucleotide by reaction with
the vacant electrophile.
[0025] If the substrate is prepared from a combination of amino-
and hydroxyl-acrylamides, the substrate can include a combination
of nucleophilic amino groups and neutral hydroxyl groups. The amino
groups can be modified with di-functional bis-electrophilic
moieties, such as a di-isocyanate or bis-NHS ester, resulting in a
hydrophilic particle reactive to nucleophiles. An exemplary bis-NHS
ester includes bis-succinimidyl C2-C12 alkyl esters, such as
bis-succinimidyl suberate or bis-succinimidyl glutarate.
[0026] Other activation chemistries include incorporating multiple
steps to convert a specified functional group to accommodate
specific desired linkages. For example, a sulfonate modified
hydroxyl group can be converted into a nucleophilic group through
several methods. In an example, reaction of the sulfonate with
azide anion yields an azide substituted hydrophilic polymer. The
azide can be used directly to conjugate to an acetylene substituted
polynucleotide or oligonucleotide via "CLICK" chemistry that can be
performed with or without copper catalysis. Optionally, the azide
can be converted to amine by, for example, catalytic reduction with
hydrogen or reduction with an organic phosphine. The resulting
amine can then be converted to an electrophilic group with a
variety of reagents, such as di-isocyanates, bis-NHS esters,
cyanuric chloride, or a combination thereof. In an example, using
di-isocyanates yields a urea linkage between the polymer and a
linker that results in a residual isocyanate group that is capable
of reacting with an amino substituted polynucleotide or
oligonucleotide to yield a urea linkage between the linker and the
polynucleotide or oligonucleotide. In another example, using
bis-NHS esters yields an amide linkage between the polymer and the
linker and a residual NHS ester group that is capable of reacting
with an amino substituted polynucleotide or oligonucleotide to
yield an amide linkage between the linker and the polynucleotide or
oligonucleotide. In a further example, using cyanuric chloride
yields an amino-triazine linkage between the polymer and the linker
and two residual chloro-triazine groups one of which is capable of
reacting with an amino substituted polynucleotide or
oligonucleotide to yield an amino-triazine linkage between the
linker and the polynucleotide or oligonucleotide. Other
nucleophilic groups can be incorporated into the particle via
sulfonate activation. For example, reaction of sulfonated particles
with thiobenzoic acid anion and hydrolysis of the consequent
thiobenzoate incorporates a thiol into the particle which can be
subsequently reacted with a maleimide substituted biomolecule to
yield a thio-succinimide linkage to the biomolecule. Thiol can also
be reacted with a bromo-acetyl group.
[0027] Alternatively, acrydite oligonucleotides can be used during
the polymerization to incorporate oligonucleotides. An exemplary
acrydite oligonucleotide can include an ion-exchanged
oligonucleotides.
[0028] Returning to FIG. 1, substrates can be incorporated into the
amplification solution along with amplification reagents, such as
enzymes including polymerase or recombinase, nucleotides (e.g. A,
T, C, G, or analogs thereof), various salts or ionic compounds, or
a combination thereof. In particular, target polynucleotides, such
as polynucleotides derived from biological sources, are included in
the amplification solution.
[0029] An emulsion is formed that includes the amplification
solution as a dispersed phase, as illustrated in 104. In
particular, the amplification solution is an aqueous solution and
can be dispersed in a hydrophobic phase, such as an oil phase. The
hydrophobic phase can include fluorinated liquids, minerals oils,
silicone oils, or any combination thereof. Optionally, the
hydrophobic phase can include a surfactant, such as a non-ionic
surfactant, such as the non-ionic surfactant described below.
[0030] The emulsion can be formed utilizing a membrane-based
mechanism, in which the aqueous amplification solution and the
continuous phase hydrophobic liquid are passed through a membrane
one or more times, forming droplets of the amplification solution
within the continuous phase hydrophobic liquid. Alternatively, the
emulsion can be formed by agitating the amplification solution in
the presence of the hydrophobic liquid. In another example, the
emulsion can be formed by repeatedly aspirating and ejecting the
amplification solution and the hydrophobic continuous phase through
a pipette tip. In a further example, droplets of the aqueous
amplification solution can be injected into a stream of the
hydrophobic liquid.
[0031] Following the emulsification of the amplification solution,
droplets of amplification solution forming a dispersed phase can
include substrates and a target polynucleotide. A portion of the
droplets can include one or more substrates and a single target
polynucleotide. Other droplets can include substrates and no
polynucleotide. Other droplets may include one or more substrates
and more than one target polynucleotides.
[0032] For example, as illustrated in FIG. 2, an aqueous
amplification solution 222 includes target polynucleotides 202 and
substrates 204. Following emulsification, some droplets 206 of the
emulsion 224 can include a single target polynucleotide and one or
more substrates. Other droplets 208 of the emulsion 224 can include
a substrate and no target polynucleotide.
[0033] Returning to FIG. 1, an amplification reaction can be
performed to provide amplified substrates, as illustrated at 106.
The conditions of the amplification reaction can depend on factors,
such as the nature of the enzymes used in the amplification
solution, the concentration of individual nucleotides, a
concentration of salts or ionic compounds, among other factors. In
an example, the amplification reaction is a polymerase chain
reaction (PCR) in which the temperature cycles multiple times in a
range of 40.degree. C. to 100.degree. C. In another example, the
amplification reaction is a recombinase polymerase amplification
(RPA). Such reactions can be performed isothermally at a
temperature in a range of 40.degree. C. to 90.degree. C. Other
amplification techniques can be used, for example, polymerase
cycling assembly (PCA), asymmetric PCR, helicase-dependent
amplification, ligation-mediated PCR, multiplex-PCR,
nanoparticle-assisted PCR, or other amplification techniques.
[0034] In an example, during amplification, template
polynucleotides including a target sequence of interest and a
segment complementary to the coupling oligonucleotide hybridize to
the coupling oligonucleotide. The coupling oligonucleotide is
extended, forming a complement to the template polynucleotide. The
template polynucleotide can further include a capture moiety useful
for binding with a separation substrate for later separation of
amplified substrates from unamplified substrates.
[0035] As a result of the amplification, dispersed phase droplets
including target polynucleotides and bead substrates produce
amplified substrates including one or more copies of the target
polynucleotide conjugated to the substrate. In contrast, droplets
including a substrate and lacking a target polynucleotide do not
produce substrates that include copies of target polynucleotides
and are referred to herein as unamplified substrates.
[0036] As illustrated at 108, the emulsion is broken to recover
amplified substrates, whereby the liquid of the dispersed phase is
separated from the continuous phase. For example, the emulsion may
be applied over a breaking solution and optionally agitated or
centrifuged. In particular, centrifuging the emulsion through a
breaking solution drives substrates into the breaking solution away
from an interface between the breaking solution and the continuous
phase liquid of the emulsion. The breaking solution can be a
hydrophilic liquid, such as an aqueous solution that includes
surfactants to assist with augmenting surface tensions and
separating the dispersed phase from the continuous phase.
[0037] In an example, the breaking solution can include one or more
surfactants having a total concentration in the range of 0.01% to
20% by weight. For example, the surfactant can be included in an
amount in a range of 0.1% to 15.0%, such as a range of 0.5% to
10.0%, a range of 0.5% to 5.0% or even a range of 0.5% to 3% by
weight. In another example, the surfactant can be included in a
total amount in a range of 5.0% to 20.0%, such as a range of 10.0%
to 20.0%, or a range of 12.0% to 18.0%.
[0038] The surfactant can be an ionic surfactant, an amphoteric
surfactant, a non-ionic surfactant, or a combination thereof. The
ionic surfactant can be an anionic surfactant. An exemplary anionic
surfactant includes a sulfate surfactant, a sulfonate surfactant, a
phosphate surfactant, a carboxylate surfactant, or any combination
thereof. An exemplary sulfate surfactant includes alkyl sulfates,
such as ammonium lauryl sulfate, sodium lauryl sulfate (sodium
dodecyl sulfate, (SDS)), or a combination thereof; an alkyl ether
sulfate, such as sodium laureth sulfate, sodium myreth sulfate, or
any combination thereof; or any combination thereof. An exemplary
sulfonate surfactant includes an alkyl sulfonate, such as sodium
dodecyl sulfonate; docusates such as dioctyl sodium sulfosuccinate;
alkyl benzyl sulfonate (e.g., dodecyl benzene sulfonic acid or
salts thereof); or any combination thereof. An exemplary phosphate
surfactant includes alkyl aryl ether phosphate, alkyl ether
phosphate, or any combination thereof. An exemplary carboxylic acid
surfactant includes alkyl carboxylates, such as fatty acid salts or
sodium stearate; sodium lauroyl sarcosinate; a bile acid salt, such
as sodium deoxycholate; or any combination thereof.
[0039] In another example, the ionic surfactant can be a cationic
surfactant. An exemplary cationic surfactant includes primary,
secondary or tertiary amines, quaternary ammonium surfactants, or
any combination thereof. An exemplary quaternary ammonium
surfactant includes alkyltrimethylammonium salts such as cetyl
trimethylammonium bromide (CTAB) or cetyl trimethylammonium
chloride (CTAC); cetylpyridinium chloride (CPC); polyethoxylated
tallow amine (POEA); benzalkonium chloride (BAC); benzethonium
chloride (BZT); 5-bromo-5-nitro-1,3-dioxane;
dimethyldioctadecylammonium chloride; dioctadecyldimethylammonium
bromide (DODAB); or any combination thereof.
[0040] An exemplary amphoteric surfactant includes a primary,
secondary, or tertiary amine or a quaternary ammonium cation with a
sulfonate, carboxylate, or phosphate anion. An exemplary sulfonate
amphoteric surfactant includes
(3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate); a
sultaine such as cocamidopropyl hydroxysultaine; or any combination
thereof. An exemplary carboxylic acid amphoteric surfactant
includes amino acids, imino acids, betaines such as cocamidopropyl
betaine, or any combination thereof. An exemplary phosphate
amphoteric surfactant includes lecithin.
[0041] In another example, the surfactant can be a non-ionic
surfactant such as a polyethylene glycol-based surfactant, an alkyl
pyrrolidine surfactant, an alkyl imidazolidinone surfactant, an
alkyl morpholine surfactant, an alkyl imidazole surfactant, an
alkyl imidazoline surfactant, or a combination thereof. In a
particular example, the polyethylene-glycol-based surfactant
includes a polyethylene-glycol ether, such as an alkylphenol
polyethoxylate. In another example, the non-ionic surfactant
includes a non-ionic fluorosurfactant, such as an ethoxylated
fluorocarbon. In a further example, the surfactant solution can
include octyl pyrrolidine.
[0042] In particular, the surfactant solution can include
combinations of such surfactants. For example, the surfactant
solution can include a combination of a non-ionic surfactant with
an anionic surfactant. In a particular example, the surfactant
solution can include a non-ionic surfactant, such as a polyethylene
glycol ether, an alkyl pyrrolidine, or a non-ionic
fluorosurfactant, and an anionic surfactant, such as a sulfate
surfactant, for example SDS. In particular, the surfactant solution
can include an ionic surfactant, such as an anionic surfactant, in
an amount in a range of 0.1% to 20.0%, such as a range of 1.0% to
15.0%, or a range of 5.0% to 15.0%, or a range of 8.0% to 12.0%. In
addition, the surfactant solution can include a non-ionic
surfactant, such as alkyl pyrrolidine (e.g., octyl pyrrolidine) in
a range of 0.01% to 10.0%, such as a range of 0.05% to 8.0%, or a
range of 1.0% to 6.0%. In another example, the surfactant solution
can include a non-ionic surfactant in a range of 0.05% to 3.0%.
[0043] In a particular example, the emulsion breaking solution
includes interference probes, such as oligonucleotides
complementary to the coupling oligonucleotide or having a sequence
similar to the coupling oligonucleotide. Such an interference probe
limits free floating polynucleotides from hybridizing with
unamplified substrates.
[0044] Referring to FIG. 2, following emulsion breaking, the
remaining aqueous solution 226 includes amplified substrates 210.
The amplified substrates 210 can include the substrate 212
conjugated to a plurality of copies of the target polynucleotide
214. The solution can also include substrates 212 that do not
include copies of target polynucleotides, referred to herein as
unamplified substrates.
[0045] Returning to FIG. 1, amplified substrates are washed and
enriched, as illustrated at 110. In an example, the substrates can
be pelletized using centrifugation and excess solution can be
decanted or drawn from above the pelletized substrates. In another
example, the amplified substrates can be attached to separation
substrates used to secure the amplified substrates while the
aqueous solution surrounding the amplified substrates is
replaced.
[0046] For example, as illustrated in FIG. 2, amplified substrates
210 can be captured by a separation substrate 220. In contrast, the
unamplified substrates 212 that do not include copies of
polynucleotides do not readily attached to the separation substrate
220. Thus, when the separation substrate 220 is secured and the
attached amplified substrates 210 are held in place, unsecured
unamplified substrates are substantially washed from the solution.
In a particular example, the separation substrates 214 are magnetic
substrates that can be secured to a container wall using a magnetic
field. The amplified substrates 210 can then be separated from the
separation substrates 220, providing a solution that has
predominantly amplified substrates and substantially fewer
unamplified substrates.
[0047] In a particular example, the amplified substrates 210 can
include a capture moiety that interacts with moieties on the
separation substrates 220. The unamplified substrates can be
substantially free of the capture moieties. For example, the
template polynucleotide can be terminated with a capture moiety.
The unamplified substrates not hybridized to a template
polynucleotide lack the capture moiety and thus, do not bind with
the separation substrate. Once the amplified substrates 210 are
separated from the unamplified substrates, the amplified substrates
210 can be separated from the separation substrate, for example, by
melting or detaching the template polynucleotide from the extended
coupling oligonucleotides conjugated to the amplified substrates
210.
[0048] Returning to FIG. 1, the enriched amplified substrates can
be loaded onto a biosensor, as illustrated at 112. Depending upon
the nature of the biosensor, the biosensor can provide a surface
onto which the amplified substrates can be attached. The surface
can be flat and optionally can include regions that are more
attractive to the amplified substrates or that are modified to
secure the amplified substrates. In another example, the biosensor
can include a surface that includes discrete sites or patterned
surfaces, such as dimples, depressions, pores, wells, ridges or
channels into which the amplified substrates align. In a further
example, as illustrated in FIG. 2, the biosensor can include a
surface structure 216 that defines a well 218 into which the
amplified substrates 210 are deposited.
[0049] As illustrated at 114 of FIG. 1, the biosensor can sense
aspects of the amplified substrate. Depending upon the nature of
the biosensor, the sensor can be utilized to detect the presence of
particular sequences within the target polynucleotide, or can be
used to sequence the target polynucleotide. For example, the
biosensor may utilize fluorescence-based sequencing-by-synthesis.
In another example, the biosensor can utilize techniques for
sequencing that include sensing byproducts of nucleotide
incorporation, such as pH or the presence of pyrophosphate or
phosphate. In a further example, the biosensor can utilize
temperature or heat detection.
[0050] Returning to FIG. 2, in an example, a well 218 of an array
of wells can be operatively connected to measuring devices. For
example, for fluorescent emission methods, a well 218 can be
operatively coupled to a light detection device. In the case of
ionic detection, the lower surface of the well 218 may be disposed
over a sensor pad of an ionic sensor, such as a field effect
transistor.
[0051] One exemplary system involving sequencing via detection of
ionic byproducts of nucleotide incorporation includes semiconductor
sequencing platforms, such as an ion-based sequencing system that
sequences nucleic acid templates by detecting hydrogen ions
produced as a byproduct of nucleotide incorporation. Typically,
hydrogen ions are released as byproducts of nucleotide
incorporations occurring during template-dependent nucleic acid
synthesis by a polymerase. Such a sequencer detects the nucleotide
incorporations by detecting the hydrogen ion byproducts of the
nucleotide incorporations. Such a sequencer can accommodate a
plurality of template polynucleotides or template complements
(e.g., extended coupling oligonucleotides) to be sequenced, each
template or extended coupling oligonucleotide disposed within a
respective sequencing reaction well in an array. The wells of the
array can each be coupled to at least one ion sensor that can
detect the release of H+ ions or changes in solution pH produced as
a byproduct of nucleotide incorporation. The ion sensor comprises a
field effect transistor (FET) coupled to an ion-sensitive detection
layer that can sense the presence of H+ ions or changes in solution
pH. The ion sensor can provide output signals indicative of
nucleotide incorporation which can be represented as voltage
changes whose magnitude correlates with the H+ ion concentration in
a respective well or reaction chamber. Different nucleotide types
can be flowed serially into the reaction chamber, and can be
incorporated by the polymerase into an extending primer (or
polymerization site) in an order determined by the sequence of the
template. Each nucleotide incorporation can be accompanied by the
release of H+ ions in the reaction well, along with a concomitant
change in the localized pH. The release of H+ ions can be
registered by the FET of the sensor, which produces signals
indicating the occurrence of the nucleotide incorporation.
Nucleotides that are not incorporated during a particular
nucleotide flow may not produce signals. The amplitude of the
signals from the FET can also be correlated with the number of
nucleotides of a particular type incorporated into the extending
nucleic acid molecule thereby permitting homopolymer regions to be
resolved. Thus, during a run of the sequencer multiple nucleotide
flows into the reaction chamber along with incorporation monitoring
across a multiplicity of wells or reaction chambers can permit the
instrument to resolve the sequence of many nucleic acid templates
or extended coupling oligonucleotides simultaneously.
[0052] Optionally during sequencing, a target polynucleotide
including an initial segment A separated from a segment B by a
target region can be copied utilizing probes complementary to A and
optionally including a capture functionality indicated by a "*".
For example, as illustrated in FIG. 3, a method 300 includes
exposing a target polynucleotide A-B to a set of complementary
probes A' and A'*. As illustrated at 304, the target polynucleotide
is copied, for example, using the A'* probe resulting in a copy of
the target polynucleotide A'-B' that includes the capture
functionality (*). The copying can be repeated multiple times using
either the A' or A'* primer.
[0053] In the example illustrated in FIG. 3, the coupling
oligonucleotide conjugated to the substrate can be similar to the B
segment of the target polynucleotide and is complementary to the B'
segment of the copy of the target polynucleotide. The copies of the
target polynucleotide can hybridize to the coupling oligonucleotide
of the substrate. The coupling oligonucleotide of the substrate can
act as a primer and can be extended to be complementary to the copy
of the target polynucleotide, thus including a segment having the
same sequence as the target sequence of the target
polynucleotide.
[0054] While the substrate at 306 is illustrated as including a
single coupling oligonucleotide, the substrate can include many
copies of the coupling oligonucleotide, such as between 1000 copies
and 30 million copies of the coupling oligonucleotide. As such, the
substrate can be amplified, i.e., the coupling oligonucleotides B
can be extended using copies of the target polynucleotide that
include the capture functionality or the capture moiety (*) or that
lack the capture functionality. Thus, the amplified substrate can
include a number of template copies of the target polynucleotide
that are hybridized to the extended coupling oligonucleotide. The
substrate can also include extended coupling oligonucleotides
hybridized to copies of the target polynucleotide that lack the
capture functionality. Alternatively, the extended coupling
oligonucleotide can hybridize to an A' or A'* primer.
[0055] The coupling oligonucleotide can have a sequence of between
5 and 100 bases, such as between 10 and 50 bases. The B segments
can have the same sequence and the same number of bases as the
coupling oligonucleotide. The B' segments can have a complementary
sequence and the same number of bases as the coupling
oligonucleotide. Alternatively, the B or B' segments can have fewer
bases and be the same or complementary to at least a portion of the
coupling oligonucleotide.
[0056] As illustrated in FIG. 4, the target polynucleotide can be
generated by providing a polynucleotide having a sequence A coupled
via a target sequence to a P segment. As illustrated at 402, and
extension probe B'-P' can be hybridized to the segment and the
extension probe can be extended. As illustrated at 404, the copy
B'P'-A' includes a segment B' that is complementary to the coupling
oligonucleotide and a segment that is complementary to the target
sequence. As illustrated at 406, an A primer can be applied to the
copy B'P'-A' and extended to provide the BP-A target sequence, as
illustrated at 408. The BP-A sequence can be used at 302 of FIG. 3
to form the template polynucleotides that hybridize to the coupling
oligonucleotide conjugated to the substrate.
[0057] Turning to FIG. 5, following amplification, the emulsion 500
includes droplets 502 in which amplification reactions can take
place. Such droplets 502 can include the target polynucleotide 506,
primer sequences A' and A'* (508 or 510) and copies 512
complementary to the target polynucleotide 506 that include the
capture functionality (*) or lack the capture functionality. In
addition, the amplification reaction takes place in the presence of
substrates. The substrates can include reaction sites 514 that
include a coupling oligonucleotide extended by hybridizing the
coupling oligonucleotide to a template polynucleotide that includes
the capture functionality or reaction sites 516 including the
extended coupling oligonucleotide hybridized to template
polynucleotides that lack the capture functionality. The droplets
502 can also include free-floating nucleotides, enzymes, and
various ionic compounds. Further, the extended coupling
oligonucleotide can hybridize to A' or A'* primers.
[0058] In addition, the emulsion 500 can include droplets 504 that
include substrates and lack a target polynucleotide. As a result of
the application, substrates 518 include the coupling
oligonucleotide that has not been extended and is not hybridized to
complements to the target polynucleotide. However, in response to
breaking of emulsion, such unamplified substrates 518 can hybridize
to free-floating complements 512 of the target polynucleotide. As
illustrated at 602 of FIG. 6, such hybridization can result in
unamplified substrates 518 including the capture functionality and
thus having the ability to attach to separation substrates,
preventing such unamplified substrates from being washed out of the
solution and competing for separation substrates with the amplified
substrates.
[0059] The emulsion breaking solution can include an interference
probe B' (604) complementary to the coupling oligonucleotide, can
include an interference probe B (606) having at least a portion of
the sequence similar to the coupling oligonucleotide, or can
include both. In a particular example, the emulsion breaking
solution can include the interference probe B (606). The
interference probe 604 is complementary to the coupling
oligonucleotide and can attached to unamplified coupling
oligonucleotides on a substrate 608 and prevent subsequent binding
of free-floating complements of the target polynucleotide. In
another example, the probe 606 includes a segment similar to the
coupling oligonucleotide that can bind with the free-floating
complements of the target polynucleotide 610 and prevent them from
hybridizing with coupling oligonucleotide sites, such as those on
unamplified substrates. Such probes 604 and 606 compete with the
free-floating complements to the target polynucleotide and the
unextended coupling oligonucleotides on the unamplified substrates
to limit the unamplified substrates from acquiring capture
functionality.
[0060] In a particular example, the amplified substrates are
enriched, separating the amplified substrates from the unamplified
substrates using a magnetic separation technique. For example, as
illustrated in FIG. 7, a method 700 includes providing an emulsion
breaking solution, as illustrated at 702. The emulsion breaking
solution can include surfactants. In particular, the emulsion
breaking solution includes interference probes complementary to the
coupling oligonucleotides on the substrates or having the same
sequence as the coupling oligonucleotides and complementary to
segments of the complements to the target polynucleotide. In an
example, the emulsion breaking solution has a composition similar
to that described above.
[0061] An emulsion is applied to the emulsion breaking solution to
recover a dispersion including the amplified substrates and
unamplified substrates, as illustrated at 704. The interference
probes can interfere with the binding interaction of free-floating
complements to the target polynucleotides and the unamplified
coupling oligonucleotides of unamplified substrates.
[0062] In particular, the capture functionality can include a
component of a pair that permits binding of the capture
functionality to an associated capture functionality on the
separation substrate. For example, the capture functionality can be
one of biotin or an avidin derivative such as streptavidin. In such
an example, substrates including extended binding sites that are
hybridized to polynucleotides including the capture functionality,
such as biotin, can bind to a magnetic substrate that includes an
opposite capturing functionality, such as streptavidin. In another
example, the capture functionality can include CLICK chemistry.
[0063] As illustrated at 706, a magnetic substrate can be applied
to the recovered dispersion. The magnetic substrates can bind with
the amplified substrates by virtue of coupling sites or coupling
oligonucleotides being hybridized to polynucleotides including
capture functionality or to any A'* primer in solution. As a result
of the competing behavior with the interference probes, few, if
any, unamplified substrates hybridize to the polynucleotides having
capture functionality. Thus, the unamplified substrates are limited
from binding to the magnetic substrates. In a particular example,
the magnetic substrates can be magnetic polystyrene substrates
including streptavidin functionality.
[0064] The amplified substrates can be separated from the
unamplified substrates, as illustrated at 706. In particular, a
magnetic field may be applied to a container, drawing the magnetic
substrates that are coupled to the amplified substrates into
contact with the wall and securing the amplified substrates and
magnetic substrates within the container. The solution can then be
washed and replaced one or more times resulting in a flushing of
components and substrates (e.g., the unamplified substrates) not
secured by the magnetic substrates.
[0065] Following separation of the amplified substrates from the
unamplified substrates, the amplified substrates can be detached
from the magnetic substrates, as illustrated at 710. In a
particular example, the template polynucleotides that are
hybridized to the amplified substrates can be melted or otherwise
separated from the extended coupling oligonucleotides. Such melting
or separation can be accomplished using a change in ionic
concentration, can be accomplished using a change in temperature,
or a combination thereof. As such, the magnetic substrates secured
within the container retain the captured template polynucleotide
having the capture functionality, while the amplified substrates
are dispersed within solution and separated from the magnetic
substrates.
[0066] The method 700 illustrated in FIG. 7 can be automated using
an automated sample handling device as illustrated in FIG. 8 and
FIG. 9. An exemplary device includes a platform 4 holding an
X.times.Y array 3 (illustrated as a one x eight strip) preloaded
with a magnetic fraction sample to be separated and any
reagents/solutions/material used in the automated process. The
device can also include an arm (e.g., a mechanical stage or arm) 11
for holding a pipette 2, and a tubing port 12 for detachable
connection to a pump (not pictured) via tubing 9. The mechanical
stage or arm may be detachably connected to a cradle 10 and may
form a hinged joint therebetween. The mechanical stage or arm may
be adapted to use the pipette to engage the array 3 to position the
sample in an IN or an OUT position such that the sample at a
separation site is either within a magnetic field, for example,
formed by magnet 5, or is sufficiently distant so as not to affect
the magnetic fraction in the sample at the separation site,
respectively.
[0067] The device can further include a receptacle for a container
to which a processed sample may be transferred. For example, the
container may be a tube (e.g., a PCR tube). The receptacle may be
placed at any suitable location in the apparatus as long as it can
be reached by the device mounted on the mechanical stage. In some
embodiments, the receptacle and the container is disposed next to
the holder for holding the device.
[0068] In various embodiments, the method can be carried out as
follows using a two dimensional array with X by Y number of sites,
where X corresponds to the number of samples to be manipulated by
one automated process and where Y number of sites is 8. Embodiments
of the automated sample handling device may be particularly well
suited for performing the following method. Positions of the array
of wells are referenced herein using alphabetic identifiers A, B,
C, D, E, F, G, or H, in which adjacent wells have consecutive
alphabetic identifiers. The apparatus can perform the following
sample manipulation sequence: 1) moving tip to Y position B then
mixing solution via multiple aspirate/dispense cycles, 2)
aspirating then transferring solution to Y position A, 3) mixing
solutions at Y position A via multiple aspirate/dispense cycles, 4)
actuating magnet, then aspirating and transferring solution to Y
position G wells (fail safe), 5) returning tip array to position A,
6) de-actuating magnet, 7) dispensing wash solution and mix via
multiple aspirate/dispense cycles, 8) actuating magnet then
aspirating solution to waste reservoir, 9) repeating wash cycle as
in steps 6, 7 and 8, 10) moving tip Y position F and aspirating,
then transferring solution to Y position A wells, and 11)
de-actuating magnet and mixing solution via multiple
aspirate/dispense cycles and transferring the solution to Y
position H. After the above steps, the user collects the processed
sample in Y position H. Alternatively, a method can be implemented
using a sample tube, a reagent well, and a disposal container in
contrast to an eight position array.
[0069] As described above, the enriched amplified substrates can be
loaded into a biosensor for determining characteristics of the
amplified substrates. In particular, the enriched amplified
substrates can be used for sequence target sequences conjugated to
the amplified substrates. For example, sequencing can include
label-free DNA sequencing, and in particular, pH-based DNA
sequencing. Substrates including DNA templates and having a primer
and polymerase operably bound are loaded into reaction chambers
(such as microwells), after which repeated cycles of
deoxynucleoside triphosphate (dNTP) addition and washing are
carried out. Such templates are typically attached as clonal
populations to the substrate, such as a microparticle, bead, or the
like, and such clonal populations are loaded into reaction
chambers. In each addition step of the cycle, the polymerase
extends the primer by incorporating added dNTP when the next base
in the template is the complement of the added dNTP. When there is
one complementary base, there is one incorporation, when two, there
are two incorporations, when three, there are three incorporations,
and so on. With each such incorporation there is a hydrogen ion
released, and collectively a population of templates releasing
hydrogen ions causing very slight changes the local pH of the
reaction chamber which is detected by an electronic sensor.
[0070] FIG. 10 diagrammatically illustrates an apparatus for
carrying out pH-based nucleic acid sequencing. Each electronic
sensor of the apparatus generates an output signal that depends on
the value of a reference voltage. In FIG. 10, housing (1000)
containing fluidics circuit (1002) is connected by inlets to
reagent reservoirs (1004, 1006, 1008, 1010, and 1012), to waste
reservoir (1020) and to flow cell (1034) by passage (1032) that
connects fluidics node (1030) to inlet (1038) of flow cell (1034).
Reagents from reservoirs (1004, 1006, 1008, 1010, and 1012) may be
driven to fluidic circuit (1002) by a variety of methods including
pressure, pumps, such as syringe pumps, gravity feed, and the like,
and are selected by control of valves (1014). Control system (1018)
includes controllers for valves (1014) that generate signals for
opening and closing via electrical connection (1016). Control
system (1018) also includes controllers for other components of the
system, such as wash solution valve (1024) connected thereto by
(1022), and reference electrode (1028). Control system (1018) may
also include control and data acquisition functions for flow cell
(1034). In one mode of operation, fluidic circuit (1002) delivers a
sequence of selected reagents (1, 2, 3, 4, or 5) to flow cell
(1034) under programmed control of control system (1018), such that
in between selected reagent flows fluidics circuit (1002) is primed
and washed, and flow cell (1034) is washed. Fluids entering flow
cell (1034) exit through outlet (1040) and are deposited in waste
container (1036). Throughout such an operation, the reactions or
measurements taking place in flow cell (1034) have a stable
reference voltage because reference electrode (1028) has a
continuous, i.e. uninterrupted, electrolyte pathway with flow cell
(1034), but is in physical contact with only the wash solution.
EXAMPLE 1
[0071] Following emulsion amplification, an emulsion is applied
over 300 microliters of one of four sample breaking solutions. The
sample breaking solutions include 10% SDS, 1% octyl-pyrrolidone,
and optionally 16 uM B primer, 32 uM B primer, or 32 uM B' primer.
Substrates recovered from the emulsion are separated using a
magnetic separation technique. As illustrated in Table 1, those
breaking solutions that included an interference probe (B primer or
B' primer) exhibited increased enriched percentage, the percentage
of recovered substrates that are amplified.
TABLE-US-00001 TABLE 1 Enrichment of Substrates Avg. Enrichment
Primer Percentage 0 uM 77.25 16 uM B primer 90 32 uM B primer 93.3
32 uM B' primer 93.9
EXAMPLE 2
[0072] Following emulsion amplification, an emulsion is applied
over 300 microliters of one of four sample breaking solutions. The
sample breaking solutions include 10% SDS, 1% octyl-pyrrolidone,
and optionally B primer in concentrations of 1.6 uM, 3.2 uM, 6.4
uM, 16 uM, or 32 uM. Substrates recovered from the emulsion are
separated using a magnetic separation technique. As illustrated in
Table 2, those breaking solutions that included an interference
probe (B primer) exhibited increased enriched percentage, the
percentage of recovered substrates that are amplified.
TABLE-US-00002 TABLE 2 Enrichment of Substrates Avg. Enrichment uM
B Primer Percentage 1.6 93.5 3.2 94.05 6.4 96.15 16 98.5 32
97.95
[0073] In a first aspect, a method of separating bead substrates
includes applying an emulsion to an emulsion-breaking solution. A
dispersed phase of the emulsion includes an unbound polynucleotide,
a first set of bead substrates and a second set of bead substrates.
The unbound polynucleotide includes a segment complementary to a
coupling oligonucleotide. The first set of bead substrates includes
the coupling oligonucleotide extended to include a segment
complementary to a portion of the unbound polynucleotide. The
second set of bead substrates includes the coupling
oligonucleotide. The emulsion-breaking solution includes an
interference probe having a sequence similar to the coupling
oligonucleotide or complementary to the coupling oligonucleotide.
The method further includes binding beads of the first set of bead
substrates to separation substrates and separating unbound beads of
the second set of bead substrates from the beads of the first set
of bead substrates bound to the separation substrates.
[0074] In an example of the first aspect, the interference probe
has the sequence similar to the coupling oligonucleotide.
[0075] In another example of the first aspect and the above
examples, the interference probe has a sequence complementary to
the coupling oligonucleotide.
[0076] In a further example of the first aspect and the above
examples, beads of the second set of bead substrates are
substantially free of extension to the coupling
oligonucleotide.
[0077] In an additional example of the first aspect and the above
examples, the unbound polynucleotide includes a capture
functionality. In an example, the extended coupling oligonucleotide
of the first set of bead substrates is hybridized to a
polynucleotide including the capture functionality. For example,
the coupling functionality includes biotin. In a further example,
the separation substrates include functionality to attach to the
capture functionality.
[0078] In another example of the first aspect and the above
examples, the separation substrates include magnetic beads. For
example, the method further includes, prior to separating the
unbound beads of the second set of bead substrates, immobilizing
with a magnet the magnetic beads bound to beads of the first set of
bead substrates.
[0079] In a further example of the first aspect and the above
examples, the emulsion-breaking solution includes a surfactant. For
example, the surfactant includes a non-ionic surfactant. In another
example, the surfactant includes an ionic surfactant. For example,
the ionic surfactant is an anionic surfactant.
[0080] In a second aspect, a method of separating bead substrates
includes applying an emulsion to an emulsion-breaking solution. A
dispersed phase of the emulsion comprising an unbound
polynucleotide, a first set of bead substrates and a second set of
bead substrates. The unbound polynucleotide includes a first
segment complementary to a coupling oligonucleotide and a second
segment attached to a capture functionality. The first set of bead
substrates includes the coupling oligonucleotide hybridized to a
polynucleotide including the first segment complementary to the
coupling nucleotide and the second segment attached to the capture
functionality. The second set of bead substrates including the
coupling oligonucleotide substantially not hybridized to the
polynucleotide or the unbound polynucleotide. The emulsion-breaking
solution includes an interference probe having a sequence similar
to the coupling oligonucleotide or complementary to the coupling
oligonucleotide. The method further includes binding beads of the
first set of bead substrates to separation substrates, the
separation substrates including functionality to attach to the
capture functionality and separating unbound beads of the second
set of bead substrates from the bound beads of the first set of
bead substrates.
[0081] In an example of the second aspect, the interference probe
has the sequence similar to the coupling oligonucleotide.
[0082] In another example of the second aspect, the interference
probe has a sequence complementary to the coupling
oligonucleotide.
[0083] In a further example of the second aspect and the above
examples, the coupling functionality includes biotin. For example,
the functionality to attach to the capture functionality includes
streptavidin.
[0084] In an additional example of the second aspect and the above
examples, the separation substrates include magnetic beads. For
example, the method further includes, prior to separating the
unbound beads of the second set of bead substrates, immobilizing
with a magnet the magnetic beads bound to beads of the first set of
bead substrates.
[0085] In another example of the second aspect and the above
examples, the emulsion-breaking solution includes a surfactant. For
example, the surfactant includes a non-ionic surfactant. In another
example, the surfactant includes an ionic surfactant. For example,
the ionic surfactant is an anionic surfactant.
[0086] In a third aspect, a kit includes a dispersion of substrates
conjugated to coupling oligonucleotides and an emulsion breaking
solution including a surfactant and an interference probe, the
interference probe having a sequence similar to the coupling
oligonucleotide or having a sequence complementary to the coupling
oligonucleotide.
[0087] In an example of the third aspect, the surfactant includes a
non-ionic surfactant.
[0088] In another example of the third aspect, the surfactant
includes an ionic surfactant. For example, the ionic surfactant is
an anionic surfactant.
[0089] In a further example of the third aspect and the above
examples, the interference probe has a sequence similar to the
coupling oligonucleotide.
[0090] In an additional example of the third aspect and the above
examples, the interference probe has a sequence complementary to
the coupling oligonucleotide.
[0091] In another example of the third aspect and the above
examples, the kit further includes a hydrophobic liquid for forming
a continuous phase of an emulsion.
[0092] Note that not all of the activities described above in the
general description or the examples are required, that a portion of
a specific activity may not be required, and that one or more
further activities may be performed in addition to those described.
Still further, the order in which activities are listed are not
necessarily the order in which they are performed.
[0093] In the foregoing specification, the concepts have been
described with reference to specific embodiments. However, one of
ordinary skill in the art appreciates that various modifications
and changes can be made without departing from the scope of the
invention as set forth in the claims below. Accordingly, the
specification and figures are to be regarded in an illustrative
rather than a restrictive sense, and all such modifications are
intended to be included within the scope of invention.
[0094] As used herein, the terms "comprises," "comprising,"
"includes," "including," "has," "having" or any other variation
thereof, are intended to cover a non-exclusive inclusion. For
example, a process, method, article, or apparatus that comprises a
list of features is not necessarily limited only to those features
but may include other features not expressly listed or inherent to
such process, method, article, or apparatus. Further, unless
expressly stated to the contrary, "or" refers to an inclusive-or
and not to an exclusive-or. For example, a condition A or B is
satisfied by any one of the following: A is true (or present) and B
is false (or not present), A is false (or not present) and B is
true (or present), and both A and B are true (or present).
[0095] Also, the use of "a" or "an" are employed to describe
elements and components described herein. This is done merely for
convenience and to give a general sense of the scope of the
invention. This description should be read to include one or at
least one and the singular also includes the plural unless it is
obvious that it is meant otherwise.
[0096] Benefits, other advantages, and solutions to problems have
been described above with regard to specific embodiments. However,
the benefits, advantages, solutions to problems, and any feature(s)
that may cause any benefit, advantage, or solution to occur or
become more pronounced are not to be construed as a critical,
required, or essential feature of any or all the claims.
[0097] After reading the specification, skilled artisans will
appreciate that certain features are, for clarity, described herein
in the context of separate embodiments, may also be provided in
combination in a single embodiment. Conversely, various features
that are, for brevity, described in the context of a single
embodiment, may also be provided separately or in any
subcombination. Further, references to values stated in ranges
include each and every value within that range.
* * * * *